1. Field of the Invention
The present disclosure relates to a domain promoting cell adhesion, spreading, migration, and neurite outgrowth and active peptides thereof, more particularly, to human laminin-2 α2 chain LG3 domain promoting cell adhesion, spreading, migration, and neurite outgrowth, and active peptides in the LG3 domain.
2. Description of the Related Art
Integrins are transmembrane adhesive receptors that mediate cell-cell and cell-extracellular matrix (ECM) interactions (Hynes, R. O. Cell, 1992). Integrin-mediated cell adhesion has been shown to regulate intracellular signaling pathways that orchestrate many cellular functions, such as proliferation, differentiation, survival, and migration (Clark, E. A., et al, Science, 1995). Signals from within the cell can modulate the integrin affinity and avidity for its ECM ligands, leading to changes in cell adhesion and migration (“inside-out” signaling) (Schwartz, M. A., et al, Annu. Rev. Cell Dev. Biol., 1995; Hynes, R. O. Cell, 1992). Conversely, the binding of integrins to ECM proteins triggers a signal transduction cascade, so-called “outside-in” signaling (Schwartz, M. A., et al, Annu. Rev. Cell Dev. Biol., 1995; Hynes, R. O. Cell, 1992), through its cytoplasmic domain that associates with focal adhesion kinase (FAK), Src-family kinases, and cytoskeleton-associated proteins, such as vinculin, talin, and paxillin (Liu, S., et al., J. Cell Sci., 2000). Engagement of integrins by ligands induces activation of a number of intracellular signaling pathways, including mitogen-activated protein kinases, Rho family GTPases, phosphatidylinositol 3-kinase (PI3K), and protein kinase C (PKC) (Schlaepfer, D. D., et al., Trends Cell Biol., 1998; Aplin, A. E., et al., Pharmacol. Rev., 1998).
PKC is an important regulator of integrin-mediated signaling and cellular behavior, such as cell adhesion, spreading, migration, and focal adhesion formation (Woods, A., et al., J. Cell Sci., 1992; Lewis, J. M., et al., J. Cell Biol., 1996). The PKC family consists of at least 12 isoforms, which can be classified into three subfamilies base upon their primary structure (Ron, D., et al., FASEB J., 1999): the conventional PKC isoforms PKCα, βI, βII and γ which are activated by calcium and diacylglycerol; the novel PKC isoforms, PKCδ, ε, η, θ, and μ that are activated by diacylglycerol but not by calcium; and the atypical PKC isoforms, PKC and t/A which are unresponsive to either diacylglycerol or calcium.
PKC activation by phorbol myristate acetate has been shown to induce the adhesion, spreading, and migration of cells (Huang, X., et al., J. Cell Sci., 1998). PKC inhibition by pharmacological reagents inhibits cell adhesion and spreading as well as focal adhesion formation and FAK phosphorylation (Woods, A., et al., J. Cell Sci., 1992; Disatnik, M. H., et al., J. Cell Sci., 2002). Several PKC isoforms have been identified in the regulation of cellular behavior. For example, PKCε activation is involved in inducing muscle cell adhesion to fibronectin, and PKCα and δ mediate cell spreading (Disatnik, M. H., et al., J. Cell Sci., 2002). PKCδ is recruited to focal adhesions in fibroblast adhesion to fibronectin, and PKCα has been localized to focal adhesions in rat embryo fibroblasts (Barry, S. T., et al., J. Cell Sci., 1994; Jaken, S., et al., J. Cell Biol., 1989). However, the mechanism of PKC activation in integrin-mediated intracellular adhesion, migration, spreading, and activities is not well understood.
Laminins are a family of basement membrane glycoproteins that has been shown to regulate a diverse array of biological activities, such as cell adhesion, spreading, migration, neurite outgrowth, tumor metastasis, angiogenesis, differentiation, and wound healing (Colognato, H., et al., Dev. Dyn, 2000). Laminins are composed of α, β, and γ polypeptide chains. The laminin α2 chain, a component of laminin-2 (α2β1γ1), laminin-4 (α2β2γ1), and laminin-12 (α2β1γ3), is expressed in skeletal and cardiac muscles, peripheral nerves, brain, and placenta (Leivo, I., et al., Proc. Natl. Acad. Sci. USA, 1988). The α chains contain a C-terminal large globular (LG) domain consisting of five globular modules (LG1-LG5) (Colognato, H., et al., Dev. Dyn, 2000). These have been known to bind integrins, α-dystroglycan, and syndecans, and be implicated as active regions for various biological functions. However, the receptor-binding motifs within LG domains of human laminin α2 chain and their biological functions and downstream signaling pathways are poorly understood.
Thus, the present inventors have performed research to identify an active motif within the human laminin α2 which is related to biological activities such as cell adhesion, spreading, migration, etc., thus identified that among five domains composing human laminin α2 chain, LG3 domain protein is involved in integrin-mediated cell adhesion, spreading, migration, and neurite outgrowth, and synthesized amino acids composing LG3 domain to measure biological activities, thus identified that a RNIPPFEGCIWN (SEQ ID NO:1) motif at position 2675 to 2686 in human laminin α2 chain is involved in promoting cell adhesion, spreading, migration, and neurite outgrowth and the integrin-mediated cell adhesion is achieved through the membrane recruitment of PKCδ and FAK phosphorylation at Tyr-397, thereby leading to completion of the present invention.